Targeting anti-cancer agents to bone using bisphosphonates

Abstract

The skeleton is affected by numerous primary and metastatic solid and hematopoietic malignant tumors, which can cause localized sites of osteolysis or osteosclerosis that can weaken bones and increase the risk of fractures in affected patients. Chemotherapeutic drugs can eliminate some tumors in bones or reduce their volume and skeletal-related events, but adverse effects on non-target organs can significantly limit the amount of drug that can be administered to patients. In these circumstances, it may be impossible to deliver therapeutic drug concentrations to tumor sites in bones. One attractive mechanism to approach this challenge is to conjugate drugs to bisphosphonates, which can target them to bone where they can be released at diseased sites. Multiple attempts have been made to do this since the 1990s with limited degrees of success. Here, we review the results of pre-clinical and clinical studies made to target FDA-approved drugs and other antineoplastic small molecules to bone to treat diseases affecting the skeleton, including osteoporosis, metastatic bone disease, multiple myeloma and osteosarcoma. Results to date are encouraging and indicate that drug efficacy can be increased and side effects reduced using these approaches. Despite these successes, challenges remain: no drugs have gone beyond small phase 2 clinical trials, and major pharmaceutical companies have shown little interest in the approach to repurpose any of their drugs or to embrace the technology. Nevertheless, interest shown by smaller biotechnology companies in the technology suggests that bone-targeting of drugs with bisphosphonates has a viable future.

Keywords: Bisphosphonate; Bone metastasis; Bone-targeting; Bortezomib; Chloroquine; Drug conjugate.

Figure 1.. Chemical structures of AP23451, alendronate and zoledronate and effects of Src inhibitor, AP23451, and alendronate on ovariectomy-induced bone loss.

(A). Chemical structures of AP23451, alendronate and zoledronate. (B). 3-m-old female mice were sham operated or ovariectomized and treated with vehicle, AP23451 or alendronate at the indicated doses once per day for 35 days. After sacrifice, bone mineral density (BMD) was measured in L2–4 lumber vertebrae using dual X-ray absorptiometry. Data are means +/− SD. One-way ANOVA with Turkey’s test. *: p<0.05 vs ovariectomy.

Figure 2.. AP23451 prevents MDA-MB-231 breast cancer-induced osteolysis and reduces, but does not eliminate tumor cell growth in long bones.

MDA-MB-231 cells (10⁴–10⁶) were inoculated into the left cardiac ventricle of 3-m-old female nude mice, which were given daily s.c. injections of AP23451 (10 mg/kg) or zoledronate (0.25mg/kg) for 28 days. After sacrifice, all femora, tibiae and humeri were removed and fixed in 10% phosphate-buffered formalin, decalcified in 14% EDTA and embedded in paraffin. (A). H&E-stained sections of distal femora showing an osteolytic lesion in the metaphysis of a vehicle-treated mouse (arrows), but not in AP23451- or zoledronate-treated mice, which developed osteopetrosis (vertical lines, where unresorbed bone largely fills the BM cavity) during the 28 days of treatment as a consequence of the inhibitory effects of these agents on bone resorption. The numbers of osteolytic metastases were counted in these 6 bones from each mouse. (B). Volume of tumor inside bones and trabecular bone volume (left panel) and volumes of tumor deposits outside bones (right panel). C. H&E-stained sections of distal femora showing replacement of metaphyseal bone and diaphyseal bone marrow by tumor cells in a vehicle-treated mouse. Osteopetrosis in the metaphysis of AP23451- and zoledronate-treated mice as well as tumor deposits (black arrows) in the BM where there are surviving unresorbed bone trabeculae (yellow arrows), and the numbers of metastases (mets) and % of metastases with osteolysis. Data are means +/− SD. One-way ANOVA with Bonferroni test. *: p<0.05 vs vehicle-treated mice.

Figure 3.. Differing effects of AP23451 and zoledronate on osteoclasts in metaphyses of mice inoculated with MDA-MB-231 breast cancer cells.

(A). H&E-stained sections of distal femora of mice showing occasional surviving (yellow arrows) osteoclasts in AP23451-treated mice and surviving and apoptotic (black arrows) osteoclasts in zoledronate-treated mice. (B). Total numbers of osteoclasts in femoral metaphyses (left panel) and % of osteoclasts with the typical features of apoptosis (cytoplasmic contraction and nuclear condensation and fragmentation). Data are means +/− SD. One-way ANOVA with Bonferroni test. * p < 0.05 vs zoledronate.

Figure 4.

Cartoon of the bone-targeting delivery concept (A) and structure and formulation of BP-Btz (A) and UR70 (B).

Figure 5.. BP-Btz has a longer half-life in bone than Btz.

3 (A) 3.5-m-old female C57BL/6 mice were given single retro-orbital intravenous injections of equimolar doses of Btz (0.6mg/kg) or BP-Btz (1.243 mg/kg). Blood was drawn at 10 min, 12, 24, and 36 hr after injection. Leg bones were harvested at sacrifice at 36 hrs. (B) Btz levels in plasma and leg bones were measured in each mouse by liquid chromatography with liquid chromatography-mass spectrometry.

Figure 6.. Bone-targeted UR70 reduces tumor burden in mice bearing Btz-resistant myeloma cells.

(A) Treatment protocol for 7-wk-old male NSG mice given injections of 0.5 × 10⁶ Btz-resistant 5TGM1-GFP mouse myeloma cells into each tibia. 3 days after tumor cell inoculation, mice were given retro-orbital intravenous injections of equimolar concentrations of the BP (0.699mg/kg), Btz (0.6mg/kg) or UR70 (1.722mg/kg) every 3 days for 8 cycles. (B) Representative images of legs from mice from each group 3 days after the last injection, visualized with an IVIS Spectrum in vivo imaging system and showing GFP-positive tumor in the injected tibiae. (C) Tumor burden assessed by GFP intensity. Unit: Average Radiant Efficiency [p/s/cm²/sr] / [μW/cm²]. Data are mean ± SD. One-way ANOVA with Turkey’s test. *: p<0.05 vs vehicle.